A DNA vaccine expressing PB1 protein of influenza A virus protects mice against virus infection.

Although influenza DNA vaccine research has focused mainly on viral hemagglutinin and has led to promising results, other virion proteins have also shown some protective potential. In this work, we explored the potential of a DNA vaccine based on the PB1 protein to protect BALB/c mice against lethal influenza A virus infection. The DNA vaccine consisted of pTriEx4 plasmid expressing PB1. As a positive control, a pTriEx4 plasmid expressing influenza A virus HA was used. Two weeks after three subcutaneous doses of DNA vaccine, the mice were challenged intranasally with 1 LD50 of A/Puerto Rico/8/34 (H1N1) virus, and PB1- and HA-specific antibodies, survival rate, body weight change, viral mRNA load, infectious virus titer in the lungs, cytokines IL-2, IL-4 and IL-10, and granzyme-B were measured. The results showed that (i) the PB1-expressing DNA vaccine provided a fair protective immunity in the mouse model and (ii) viral structural proteins such as PB1 represent promising antigens for DNA vaccination against influenza A.

Antibodies to PB1-F2 protein are induced in response to influenza A virus infection.

PB1-F2 is a small influenza A virus (IAV) protein encoded by an alternative (+1) reading frame of the PB1 gene. While dispensable for IAV replication in cultured cells, PB1-F2 has been implicated in IAV pathogenicity. To better understand PB1-F2 expression in vivo and its immunogenicity, we analyzed anti-PB1-F2 antibodies (Abs) in sera of mice infected intranasally (i.n.) with A/PR/8/34 (H1N1) virus and human acute and convalescent sera collected from the influenza H3N2 winter 2003-2004 epidemic. We explored a number of methods for detecting anti-PB1-F2 Abs, finding that PB1-F2-specific Abs could clearly be detected via immunoprecipitation or immunofluorescence assays using both immune mouse and human convalescent sera. Importantly, paired human sera exhibited similar increases in HI titers and PB1-F2-specific Abs. This study indicates that PB1-F2 is expressed in sufficient quantities in mice and humans infected with IAV to elicit an Ab response, supporting the biological relevance of this intriguing accessory protein.

The ubiquitination of the influenza A virus PB1-F2 protein is crucial for its biological function.

The aim of the present study was to identify what influences the short half-life of the influenza A virus PB1-F2 protein and whether a prolonged half-life affects the properties of this molecule. We hypothesized that the short half-life of PB1-F2 could conceal the phenotype of the protein. Because proteasome degradation might be involved in PB1-F2 degradation, we focused on ubiquitination, a common label for proteasome targeting. A cluster of lysine residues was demonstrated as an ubiquitination acceptor site in evolutionary and functionally distinct proteins. The PB1-F2 sequence alignment revealed a cluster of lysines on the carboxy terminal end of PB1-F2 in almost all of the GenBank sequences available to date. Using a proximity ligation assay, we identified ubiquitination as a novel posttranslational modification of PB1-F2. Changing the lysines at positions 73, 78, and 85 to arginines suppressed the ubiquitination of A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. The mutation of the C-terminal lysine residue cluster positively affected the overall expression levels of avian A/Honk Kong/156/1997 (H5N1)- and mammalian A/Puerto Rico/8/1934 (H1N1)-derived PB1-F2. Moreover, increased PB1-F2 copy numbers strengthened the functions of this virus in the infected cells. The results of a minigenome luciferase reporter assay revealed an enhancement of viral RNA-dependent RNA polymerase activity in the presence of stabilized PB1-F2, regardless of viral origin. IFNß antagonism was enhanced in 293T cells transfected with a plasmid expressing stabilized K→R mutant variants of PB1-F2. Compared with PB1-F2 wt, the loss of ubiquitination enhanced the antibody response after DNA vaccination. In summary, we revealed that PB1-F2 is an ubiquitinated IAV protein, and this posttranslational modification plays a central role in the regulation of the biological functions of this protein.

The 4H84 monoclonal antibody detecting beta2m free nonclassical HLA-G molecules also binds to free heavy chains of classical HLA class I antigens present on activated lymphocytes.

We have found that 4H84 monoclonal antibody (mAb) used for detection of beta2m free HLA-G molecules also binds to free heavy chains of classical HLA class I antigens generated on the cell surface by mild acid treatment. Here we demonstrate that beta2m free classical HLA class I molecules induced on the surface of activated lymphocytes not expressing HLA-G also bind 4H84 mAb. These results demonstrate that 4H84 mAb should be used for detection of HLA-G in cells and tissues with backing by other HLA-G specific mAbs.

Mild acid treatment induces cross-reactivity of 4H84 monoclonal antibody specific to nonclassical HLA-G antigen with classical HLA class I molecules.

Mild acid treatment by releasing beta(2)m and antigenic peptides leaves human leukocyte antigen (HLA) class I free heavy chains attached to the cell surface. Acid treatment thus allows detection of the cell surface class I antigens by monoclonal antibodies (mAbs) specific to HLA-free heavy chains. We found that acid treatment also enables detection of the cell surface non-classical HLA-G class I antigen with mAbs specific for HLA-G free heavy chains, including 4H84 mAb recognizing all isoforms. Furthermore, we found that 4H84 mAb, but not other mAbs specific to HLA-G free heavy chains, binds to the surface of 8 out of 16 acid-treated leukemia cell lines. Nevertheless, HLA-G antigen is not present in any of these leukemia cells. This was demonstrated by failure to detect any antigen with 4H84 mAb in immunoblotting as well as by inability to detect HLA-G mRNA by RT-PCR. The antigen recognized by 4H84 mAb in some acid treated leukemia cells was identified by immunoprecipitation as a 45 kDa protein. A number of observations indicate that 45 kDa proteins are none other than classical class I heavy chains. Acid treatment thus induces the ability of the 4H84 mAb to recognize some classical HLA class I molecules. Remarkably, 4H84 determinant on HLA-G is linear but corresponding determinant present on some partially folded classical HLA class I free heavy chains is conformational. In view of the unexpected cross-reactivity, detection of HLA-G with this mAb must be carefully evaluated to avoid false detection.

Analysis of HLA-G expression in malignant hematopoetic cells from leukemia patients.

It has been suggested that HLA-G antigens may provide tumor cells with an effective immune escape mechanism. So far mostly solid tumors have been analyzed; HLA-G antigen was only exceptionally detected. To further examine HLA-G expression, patients were chosen with different forms of leukemia: AML (25), CML (4), ALL (9), CLL (8), HCL (2) and NHL (3). Using flow cytometry with three HLA-G specific mAbs (87G, 01G and MEM-G/9), western blotting with two specific mAbs (4H84 and MEM-G/1) and RT-PCR, neither HLA-G antigen nor mRNA for any HLA-G isoform was detected. These results strongly suggest that HLA-G antigen is not expressed in freshly isolated human leukemia cells and therefore is not involved in their escape from immune attack.

HLA-G5 in the blood of leukemia patients and healthy individuals.

In this work we focused on analysis of HLA-G5 molecules in the blood of patients with B-CLL leukemia and healthy individuals. Using sandwich ELISA, we found total soluble HLA-G, represented by sHLA-G1 and HLA-G5 in most of B-CLL patients while HLA-G5 alone was present only in few cases in both groups. These results lead us to assume that the majority of soluble HLA-G in blood is generated by proteolytic cleavage and shedding of membrane-bound HLA-G1. There is no correlation between the presence of HLA-G5 in blood of B-CLL patients and the stage of disease, age, and gender.

Defining influenza A virus hemagglutinin antigenic drift by sequential monoclonal antibody selection.

Human influenza A virus (IAV) vaccination is limited by "antigenic drift," rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines.

Development of a quantitative cell-based intracellular ELISA for the screening of B cell hybridoma supernatants: a novel rapid assay to detect positive clones.

The primary screening of hybridoma clones secreting monoclonal antibodies (MAbs) requires the testing of a large number of hybridoma culture supernatants within a short time and is very labor-intensive. In addition, the type of antigen and its location in the cell have to be considered when selecting the appropriate screening procedure, but relatively few reagents are available for analyzing these molecules. We have developed an intracellular and cell surface ELISA technique for screening hybridoma supernatants that hastens the screening procedure of a large number of clones in a short period of time, as the supernatants of fused cells grown in 96-well plates are used directly in the assay. This novel screening technique is rapid, sensitive, specific, and applicable to MAbs specific for a wide variety of intracellular and/or cell surface proteins.